Stabilized thrombin compositions

ABSTRACT

Stabilized thrombin compositions, processes for preparing them, and kits comprising them are disclosed. The compositions comprise thrombin, a bacteriostatically effective amount of benzyl alcohol or chlorobutanol, and 0.10%-5.0% (w/v) sucrose in aqueous solution. The compositions are stable when stored at 2° C.-8° C. for four weeks or more.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/944,224, filed Jun. 15, 2007, which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

Formulation of pharmaceutical proteins presents significant challenges.Proteins possess multiple functional groups in addition tothree-dimensional structure; degradation therefore proceeds via bothchemical (modifications involving bond formation or cleavage) andphysical (denaturation, aggregation, adsorption, precipitation)pathways. Since each protein embodies a unique combination of amino acidsequence, isoelectric point, and other determinants, its response tochanges in solution conditions is unpredictable, and must be determinedon a case-by-case basis. Attempts to prevent one form of degradationoften increase the rate of another.

Degradation of proteins can be greatly reduced or avoided bylyophilization. However, lyophilization is time consuming and costly,and can cause protein denaturation and aggregation if appropriateexcipients are not included. As with solution formulation, stabilizationof lyophilized proteins must be dealt with on an individual basis.Sodium chloride can be used to maintain stability during purificationand storage by reducing aggregation and precipitation. However, sodiumchloride is a problematic excipient in lyophilized formulations becauseit lowers glass transition temperature, thereby necessitating lowprimary temperatures and long cycle times. Jiang et al, US 20060002918A1 disclose methods for stabilizing lyophilized thrombin preparations byformulating the thrombin with sucrose, mannitol, sodium chloride, and asurfactant or high molecular weight polyethylene glycol. However,stabilization of a protein in the lyophilized state does not ensurestability upon reconstitution, and protein solutions must often bediscarded if not used promptly after reconstitution. In addition, thein-use handling of a lyophilized drug product is often limited bysterility considerations because the container-closure integrity iscompromised during reconstitution. As such, the in-use handling timecould be extended if a drug product formulation was stable in thepresence of a preservative.

In view of the often high cost of protein therapeutics, means ofenhancing the stability of these proteins in solution are needed. In thecase of thrombin, various excipients have been proposed for stabilizingthe protein. For example, Silbering et al., U.S. Pat. No. 4,696,812disclose the use of low levels of saline and glycerol to reducedenaturation of thrombin in solution. Nishimaki et al., U.S. Pat. No.5,130,244 disclose an aqueous thrombin solution containing a sugar andan amino acid as stabilizers.

Despite these advances, there remains a need in the art for preparationsof thrombin that can be stored for extended periods of time in liquidform.

SUMMARY OF THE INVENTION

The present invention provides storage-stable compositions of thrombinand related processes and kits. Thrombins for use within the inventioninclude, without limitation, human thrombin, recombinant human thrombin,and bovine thrombin.

Within one aspect of the invention there is provided a pharmaceuticalcomposition comprising thrombin, a bacteriostatically effective amountof a preservative selected from the group consisting of benzyl alcoholand chlorobutanol, and 0.10% to 5.0% (w/v) sucrose in aqueous solution.Within certain embodiments, the composition further comprises one ormore of a buffer, a salt, a polyol, a surfactant, an amino acid, or anadditional carbohydrate. Within another embodiment, the preservative isbenzyl alcohol. Within a related embodiment, the preservative is benzylalcohol at a concentration of 0.8%-1.5%, v/v. Within another embodiment,the composition further comprises mannitol. Within a related embodiment,mannitol and sucrose are present in the composition at a ratio ofmannitol:sucrose greater than 1:1 but not greater than 2.5:1 (w/w).

Within a second aspect of the invention there is provided a compositioncomprising thrombin, a pharmaceutically acceptable buffer, 0.10% to 5%(w/v) sucrose, and a preservative selected from the group consisting ofbenzyl alcohol at a concentration of 0.8% to 1.5% (v/v) or chlorobutanolat a concentration of 0.4% to 0.6% (w/v), in aqueous solution at pH5.7-7.4. Within one embodiment, the preservative is benzyl alcohol.Within a related embodiment, the preservative is benzyl alcohol at aconcentration of 0.8%-1.0%, v/v. Within other embodiments the sucroseconcentration is 0.5% to 3.0% (w/v) or about 1% (w/v). Within otherembodiments the buffer is selected from the group consisting ofhistidine, citrate, phosphate, Tris, succinate, and acetate buffers.Within another embodiment, the composition further comprises mannitol.Within a related embodiment, mannitol and sucrose are present in thecomposition at a ratio of mannitol:sucrose greater than 1:1 but notgreater than 2.5:1 (w/w). Within another embodiment, the diluentcomprises sodium chloride. Within further embodiments, the thrombin ispresent at a concentration of 0.1 mg/mL to 5.0 mg/mL or a concentrationof 0.3 mg/mL to 3.0 mg/mL.

Within a third aspect of the invention there is provided an aqueouscomposition consisting essentially of 0.03 mg/mL to 1.6 mg/mL thrombin,0.17% to 1.3% (w/v) sucrose, 1.1% to 1.6% (w/v) mannitol, 0.8% to 2.0%(w/v) NaCl, 0-1.6 mM CaCl₂, 0.001% to 0.32% (w/v) of a surfactant orhigh-molecular-weight polyethylene glycol, a pharmaceutically acceptablebuffer, and a bacteriostatically effective amount of a preservativeselected from the group consisting of benzyl alcohol and chlorobutanolin aqueous solution at pH 5.7-7.4. The concentration of the buffer isselected to provide approximately physiological pH upon application ofthe composition in a surgical setting, and the ratio of mannitol:sucroseis greater than 1:1 but not greater than 2.5:1 (w/w). Within oneembodiment the ratio of mannitol to sucrose is 1.33:1 (w/w). Withinother embodiments the molar ratio of sucrose:thrombin is at least 700:1or at least 2000:1. Within another embodiment the preservative is benzylalcohol at a concentration of 0.8% to 1.5% (v/v).

Within a fourth aspect of the invention there is provided a process forpreparing a stabilized thrombin solution comprising the steps of (a)providing a lyophilized composition comprising thrombin and a quantityof sucrose sufficient to stabilize the protein in the presence of abacteriostatically effective amount of a preservative selected from thegroup consisting of benzyl alcohol and chlorobutanol, (b) providing adiluent comprising a bacteriostatically effective amount of benzylalcohol or chlorobutanol in water, and (c) combining the lyophilizedcomposition and the diluent to form a solution, wherein theconcentration of sucrose in the solution is from 0.10% to 5.0% (w/v).Within certain embodiments the lyophilized composition further comprisesa buffer, a salt, or a polyol.

Within a fifth aspect of the invention there is provided a process forpreparing a stabilized thrombin solution comprising the steps of (a)providing a lyophilized composition comprising thrombin and at least onepharmaceutically acceptable excipient, and (b) reconstituting thelyophilized composition in a diluent to provide a pharmaceuticallyacceptable thrombin solution, wherein the diluent is selected to providein the solution a benzyl alcohol concentration of 0.8% to 1.5% (v/v) anda sucrose concentration of 0.10% to 5% (w/v). Within one embodiment, thesolution further comprises mannitol. Within a related embodiment, thesolution comprises mannitol and the mannitol and sucrose are present inthe solution at a ratio of mannitol:sucrose greater than 1:1 but notgreater than 2.5:1 (w/w).

Within a sixth aspect of the invention there is provided a kitcomprising (a) a pharmaceutically acceptable diluent comprising abacteriostatically effective amount of benzyl alcohol or chlorobutanolin water in a first sealed container, and (b) a lyophilized compositioncomprising thrombin and a quantity of sucrose in a second sealedcontainer, wherein the quantity of sucrose is selected to provide asucrose concentration of 0.10% to 5.0% (w/v) upon reconstitution of thelyophilized composition with the diluent. Within one embodiment thediluent further comprises NaCl. Within a related embodiment the diluentis bacteriostatic saline. Within another embodiment, the kit furthercomprises means for transferring the diluent from the first sealedcontainer to the second sealed container. Within further embodiments,the kit comprises an instruction sheet and/or an applicator device, suchas a syringe or sprayer. Within an additional embodiment, the first andsecond sealed containers are packaged in a third container.

Within a seventh aspect of the invention there is provided a process ofadministering thrombin to a mammal in need thereof, comprising the stepsof (a) providing a pharmaceutically acceptable diluent comprising abacteriostatically effective amount of a preservative selected from thegroup consisting of benzyl alcohol and chlorobutanol, (b) providing alyophilized composition comprising thrombin and an amount of sucrose,(c) reconstituting the lyophilized composition with the diluent to forma solution, and (d) delivering the solution to the mammal, wherein theamount of sucrose is selected to provide a sucrose concentration of0.10% to 5.0% (w/v) upon reconstitution of the lyophilized compositionwith the diluent. Within one embodiment the diluent is bacteriostaticsaline.

These and other aspects of the invention will become evident uponreference to the following description of the invention.

DESCRIPTION OF THE INVENTION

“Bacteriostatic saline” is 0.9% sodium chloride in water (normal saline)containing 0.9% benzyl alcohol as a preservative.

A “bacteriostatically effective amount of a preservative” is an amountthat provides a bacteriostatic effect according to the criteria of USP51, that is an amount sufficient to protect a dosage form of atherapeutic agent from microbiological growth or from microorganismsthat are inadvertently introduced during or subsequent to manufacturing.A typical bacteriostatic range for benzyl alcohol is 0.9%-2.0% (v/v),although amounts up to 5% may be used in certain applications. A typicalbacteriostatic range for chlorobutanol is 0.4% to 0.6% (w/v). Thoseskilled in the art will recognize that the required concentration of apreservative will vary somewhat with the composition of the solution;common pharmaceutical excipients may increase or decrease bacteriostaticactivity. The actual concentration required for any solution can bedetermined according to standard testing procedures.

As used herein, a “diluent” is a solution that dilutes or renders lesspotent a therapeutic agent. The term is used to include such solutionsthat are used to reconstitute dried (e.g., lyophilized) therapeuticagents. A “pharmaceutically acceptable diluent” is a diluent that is onthe GRAS (generally recognized as safe) list as recognized bypharmaceutical regulatory bodies. Commonly used pharmaceuticallyacceptable diluents include sterile water USP, normal saline USP, and 5%dextrose USP.

As used herein, “thrombin” denotes the activated enzyme, also known as□-thrombin, which results from the proteolytic cleavage of prothrombin(factor II). As disclosed below, thrombin can be prepared by a varietyof methods known in the art, and the term “thrombin” is not intended toimply a particular method of production. Human thrombin is a 295 aminoacid protein composed of two polypeptide chains joined by a disulfidebond. Both human and non-human (e.g., bovine) thrombins can be usedwithin the present invention. Thrombin is used medically as a hemostaticagent and as a component of tissue adhesives.

Human and non-human thrombins are prepared according to methods known inthe art. Purification of thrombin from plasma is disclosed by, forexample, Bui-Khac et al., U.S. Pat. No. 5,981,254. Purification ofthrombin from plasma fractions, such as Cohn fraction III, is disclosedby Fenton et al., J. Biol Chem. 252:3587-3598, 1977. Recombinantthrombin can be prepared from a prethrombin precursor by activation witha snake venom activator as disclosed in U.S. Pat. No. 5,476,777.Suitable venom activators include ecarin and prothrombin activator fromOxyuranus scutellatus. Other activators, such as factor Xa, can also beemployed.

Highly purified thrombin has a specific activity of approximately 3200NIH Units per mg or 3800 International Units per mg. One NIH Unit equals1.19 International Unit. The abbreviation “U” is used herein to denote“Units” and indicates NIH units unless specified otherwise. Theabbreviation “IU” is used to denote International Units.

Numerical ranges (e.g., “from X to Y”) include the endpoints unlessotherwise specified.

The terms “about” and “approximately” denote a range of error of ±10% ofthe stated value. For example, “about 1%” is used to denote a range of0.9% to 1.1%, inclusive.

All references cited herein are incorporated by reference in theirentirety.

The present invention provides methods for stabilizing thrombin insolution. The inventors have found that a previously disclosed (Jiang etal., US 20060002918 A1) lyophilized formulation of recombinant humanthrombin, when reconstituted with bacteriostatic saline, remains stableat refrigerator temperature for up to thirteen weeks. This result wasunexpected in view of the known tendency of preservatives such as benzylalcohol to denature proteins and increase aggregation rates (e.g., Guptaand Kaisheva, AAPS Pharm Sci 5(2):E8, 2003; Maa and Hsu, Int. J. Pharm.140:155-168, 1996; Lam et al., Pharm. Res. 14:725-729, 1997). When apreparation of bovine thrombin (THROMBIN-JMI, King Pharmaceuticals,Inc.) was reconstituted with bacteriostatic saline, the solution becamehazy. It was discovered that the haziness of the latter preparationcould be avoided by adding sucrose to the diluent used to reconstituteit.

Compositions of the present invention comprise, in aqueous solution,thrombin, a bacteriostatically effective amount of a preservativeselected from the group consisting of benzyl alcohol and chlorobutanol,and sucrose to stabilize the thrombin against degradation induced by thepreservative. The concentration of sucrose in the composition is from0.10% to 5.0% (w/v), often 0.17% to 5.0%, usually 0.17% to 3.0%, morecommonly 0.5% to 2.0%. Within certain embodiments of the invention theconcentration of sucrose is 1% (w/v). The thrombin compositions arestable at refrigerator temperatures for at least four weeks. Recombinanthuman thrombin solutions of this type have been found to remain stablewhen tested after six and thirteen weeks of storage at 2° C.-8° C.

As will be evident to those skilled in the art, the compositions maycomprise additional components, such as buffers, bulking agents,lyoprotectants, solubilizers, surfactants, carbohydrates, polyols, aminoacids, additional carbohydrates (e.g., trehalose), and/or tonicityadjusting agents (e.g., salts, mannitol). Selection of actual componentsis a matter of routine design choice and is within the level of ordinaryskill in the art. See for example, Remington: the Science and Practiceof Pharmacy, 21st ed., Lippincott Williams & Wilkins, Baltimore, 2005.

Thrombin is commonly provided as a lyophilized powder or cake that canbe reconstituted by adding a pharmaceutically acceptable diluent andmixing. The diluent will ordinarily contain the preservative (e.g.,benzyl alcohol or chlorobutanol). The diluent volume is selected toprovide a solution having a final sucrose concentration of 0.10% to 5.0%(w/v). Sucrose may be present in the lyophilized thrombin formulation,the diluent, or both, so long as the total concentration of sucrose inthe reconstituted solution is within the range of 0.10% to 5.0%, 0.17%to 5.0%, 0.17% to 3.0%, or 0.5% to 2.0% (w/v). The lyophilizedformulation may also contain a buffer as disclosed in more detail below.In one embodiment, the formulation contains mannitol as a bulking agent.In preferred formulations, mannitol is included at a ratio ofmannitol:sucrose greater than 1:1 but not greater than 2.5:1 (w/w). Theactual selection of the diluent will be made in view of the compositionof the lyophilized formulation and the intended use of the product.However, the invention is not limited by the manner in which thecomposition is made or the order in which its constituents are combined.For example, a lyophilized formulation of thrombin containing one ormore lyoprotectants can be reconstituted with a pharmaceuticallyacceptable diluent containing the preservative and sucrose, andoptionally containing additional components as disclosed above. If longterm storage is not required or stability is augmented by storage attemperatures below freezing (<0° C.), purified thrombin can beformulated with the preservative, the sucrose, and optionally othercomponents, without preparing a lyophilized formulation.

Any pharmaceutically acceptable buffer may be employed within thepresent invention. It is preferred to select a buffer that provides aslightly acidic to approximately neutral pH to limit loss of thrombinactivity due to autolysis (formation of inactive autolytic degradationproducts including β- and γ-thrombin), which increases above pH 6.0, andprecipitation, which increases below pH 6.0. It is thereforeadvantageous to use a weak buffer that is effective at slightly acidicto approximately neutral pH (i.e, pH 5.7-7.4), but will allow the pH toreach physiological pH (i.e. pH 7.35-7.45) when the thrombin is appliedto bleeding tissue, thereby providing optimal biological activity.Thrombin compositions buffered to a pH of 6.0-6.5 are preferred.Examples of suitable buffers include histidine, phosphate, citrate, Tris(2-Amino-2-(hydroxymethyl)-1,3-propanediol), and succinate buffers. Forexample, histidine buffer can be included at a concentration of 1-20 mM,usually 2-10 mM, and more often about 5 mM.

Suitable concentrations of other buffers for use within the presentinvention can be readily determined by one of ordinary skill in the art.Formulation buffers can be tested by adding blood and measuring the pHof the resulting solution. In an exemplary assay, aliquots of rabbitblood are added stepwise to various buffers, and pH is measured aftereach addition. When histidine buffers are tested in such an assay, 3.2mM, 5 mM, 12.8 mM, and 20 mM histidine are neutralized by the additionof not more than 1.0 volume of blood. In contrast, 160 mM succinatebuffer and 90 mM phosphate/12.8 mM histidine buffer did not produce amixture with a pH above 7.0 even after addition of 2-3 volumes of blood.Thus, when buffers such as succinate or phosphate/histidine are used,lower concentrations are preferred.

Typical thrombin compositions within the present invention consistessentially of 0.03 mg/mL to 1.6 mg/mL thrombin, 0.17% to 1.3% (w/v)sucrose, a bacteriostatically effective amount of benzyl alcohol orchlorobutanol, 1.1% to 1.6% (w/v) mannitol, 0.8% to 2.0% (w/v) NaCl,0-1.6 mM CaCl₂, 0.001% to 0.32% (w/v) of a surfactant (e.g.,polyethylene oxides, sorbitan esters, polyoxyethylene alkyl ethers,glycerides of fatty acids, or polyoxyethylene sorbitan fatty acidesters) or high-molecular-weight polyethylene glycol (e.g., PEG 400, PEG1000, PEG 3350, PEG 5000, or PEG 8000), and a pharmaceuticallyacceptable buffer at pH 5.7-7.4. In certain preferred compositions, theratio of mannitol:sucrose is greater than 1:1 but not greater than 2.5:1(w/w).

The concentration of thrombin within the compositions of the presentinvention can be varied, depending on the intended use, by adjusting thedilution volume. For topical application to control bleeding, thethrombin concentration will generally be from about 100 U/mL to about20,000 U/mL (approximately 0.026 mg □-thrombin per mL to 5.26 mg□-thrombin per mL), more commonly about 300 U/mL to about 5,000 U/mL,typically about 1,000 U/mL, although the actual concentration will bedetermined by the physician according to the needs of the individualpatient. The thrombin solution can be used immediately, held at roomtemperature for up to 48 hours, or stored under refrigeration for up tofour weeks or more. The thrombin can be applied to bleeding tissue toachieve hemostasis, often in combination with an absorbable gelatinsponge or other delivery vehicle according to conventional surgicalpractice. The thrombin can also be used as a component of a tissueadhesive or fibrin glue. These and other uses of thrombin are known inthe art.

The invention further provides thrombin in a kit comprising a diluent ina first sealed container and lyophilized thrombin in a second sealedcontainer. The diluent comprises a bacteriostatically effective amountof benzyl alcohol or chlorobutanol. In typical embodiments the diluentis normal saline (0.9% w/v sodium chloride solution) containing thebenzyl alcohol or chlorobutanol. The kit may further comprise anapplication device, such as a sprayer, syringe, or the like, as well asone or more needles or other transfer devices to facilitate transfer ofliquid into and out of the containers. A variety of such devices,including needleless transfer devices, are known in the art. See, forexample, U.S. Pat. No. 6,558,365. An instruction sheet may also bepackaged in the kit. Commonly, the first and second sealed containerswill be packaged in a third container. The contents of the kit willordinarily be sterile. The third container may also be sterile.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1

Recombinant human thrombin (rhThrombin) was formulated at 1 mg/mL (3200U/mL) in 5 mM histidine, 150 mM NaCl, 4 mM CaCl₂, 0.1% PEG3350, pH 6.0with varying concentrations of mannitol and sucrose as shown in Table 1.

TABLE 1 Formulation Mannitol Sucrose T 5% 0.5%   A 5% 1% B 5% 2% C 5% 3%D 4% 3% E 3% 3% F — 5%

1.6-mL aliquots of the solutions were placed in vials and lyophilizedunder the conditions shown in Table 2. For subsequent analysis,lyophilized samples were reconstituted with water where necessary.Visual observations of lyophilized and reconstituted samples are shownin Table 3.

TABLE 2 Primary Secondary Formulation Freezing & Annealing Drying DryingT 0.5° C./min to 5° C., hold 0.5 hr 0.5° C./min to 0.2° C./min to 0.25°C./min to −50° C., hold 2 hr −10° C., hold 16 hr 30° C., hold 24 hr0.25° C./min to −30° C., hold 3 hr 60 mTorr 60 mTorr 0.25° C./min to−50° C., hold 2 hr A, B, C, D, 0.5° C./min to 5° C., hold 2 hr 0.5°C./min to −30° C., 0.5° C./min to 25° C., E, F 0.5° C./min to −50° C.,hold 2 hr hold 10 hr hold 24 hr 0.25° C./min to −20° C., hold 2 hr 0.5°C./min to −25° C., 0.5° C./min to 30° C., 0.25° C./min to −25° C., hold2 hr hold 10 hr hold 8 hr 0.25° C./min to −50° C., hold 2 hr 0.5° C./minto −20° C., 0.5° C./min to 20° C., hold 10 hr hold 6 hr 0.5° C./min to−15° C., 60 mTorr hold 10 hr 60 mTorr

TABLE 3 Formulation Lyophilized Solid Reconstituted Solution T Whitecake Clear A White cake Clear B White cake Clear C White cake Clear DWhite cake Clear E White semi-collapsed Clear cake or white powder FWhite powder Clear

Example 2

Recombinant human thrombin was formulated at 1 mg/mL in 5 mM histidine,3% (w/v) sucrose, 4% (w/v) mannitol, 150 mM NaCl, 4 mM CaCl₂, 0.1%PEG3350, pH 6.0. 1.6-mL aliquots of the solution were placed in vialsand lyophilized under the conditions shown in Table 4.

TABLE 4 Freezing & Annealing Primary Drying Secondary Drying 0.5° C./minto 5° C., hold 2 hr 0.5° C./min 0.5° C./min to 0.25° C./min to −52° C.,hold 4 hr to −10° C., 30° C., 0.5° C./min to −25° C., hold 2 hr hold 26hr; hold 24 hr; 0.5° C./min to −52° C., hold 4 hr 60 mTorr 60 mTorr

Example 3

One vial (5,000 IU) each of recombinant human thrombin (“rhThrombin DP,”prepared as disclosed in Example 2) and bovine thrombin (THROMBIN-JMI,Jones Pharma Incorporated, Bristol, Va.; lyophilized powder containingmannitol and sodium chloride) were reconstituted with 5 mL ofbacteriostatic saline USP. The bovine thrombin appeared colorless, hazyand frothy upon reconstitution, while the recombinant thrombin wascolorless and clear. Following storage at 25° C. for 48 hours, thebovine thrombin was colorless with a visible precipitate, and therecombinant thrombin remained colorless and clear.

Example 4

One vial (5,000 IU) each of rhThrombin DP, recombinant human thrombinpilot formulation (Example 1, formulation T), and bovine thrombin(THROMBIN-JMI) were reconstituted with 5.0 mL of bacteriostatic salineUSP. Sucrose concentrations in the reconstituted solutions were 0% inthe bovine thrombin, 0. 17% in the pilot thrombin, and 1.0% in therhThrombin DP. Both of the rhThrombin solutions were clear, while thebovine thrombin solution appeared hazy.

Example 5

Vials of rhThrombin DP (5,000 IU) were reconstituted with 5 mLbacteriostatic saline injection, USP (n=3). The reconstituted sampleswere stored inverted at 2-8° C. for 1, 2, 4, 6, 9, and 13 weeks, and at25° C. for 8, 24, and 48 hours. After storage, the samples were analyzedfor appearance, content (by RP-HPLC), purity (RP-HPLC), potency(clotting activity), and high-molecular-weight (HMW) impurities(SE-HPLC). As shown in Table 5, no remarkable instabilities wereobserved for the time periods and conditions that were evaluated.

TABLE 5 Clotting Specific Content Purity HMW Activity Activity TimePoint (mg/mL) (%) (%) (IU/mL) (IU/mg) 25° C. (n = 3) Initial 0.34 91.3<0.2 1254 3724  8 hr 0.33 91.7 <0.2 1261 3784 24 hr 0.33 92.1 <0.2 12453735 48 hr 0.33 92.5 <0.2 1226 3679 2-8° C. (n = 3) 1 week 0.33 91.9<0.2 1170 3582 2 week 0.32 91.8 <0.2 1164 3601 4 week 0.33 91.7 <0.21182 3546 6 week 0.33 92.2 <0.2 1243 3766 9 week 0.33 92.1 <0.2 11363479 13 week  0.32 91.9 <0.2 1102 3443

Example 6

Vials of rhThrombin DP and pilot formulation (5,000 IU each) werereconstituted with 1.6 mL of bacteriostatic saline. Sucroseconcentration was 3.0% in the rhThrombin DP solution and 0.5% in thepilot thrombin solution. No precipitation was observed right afterreconstitution or after 24 or 48 hours at 25° C.

Example 7

Three vials of recombinant human thrombin DP (5,000 IU) were eachreconstituted with 5 mL bacteriostatic saline. No precipitation wasobserved after 13 weeks of storage at 2-8° C.

Example 8

Recombinant human thrombin DP was reconstituted with 0.9% or 2.0% benzylalcohol in normal saline. Samples were stored at 25° C. No precipitationwas observed in the 0.9% samples immediately after reconstitution orafter 48 hours. Samples containing 2.0% benzyl alcohol showed noprecipitation immediately after reconstitution, but showed precipitationat 24 hours.

Example 9

Bovine thrombin was reconstituted with bacteriostatic saline containing1% sucrose. Samples were stored at 25° C. No precipitation was observedimmediately after reconstitution or after 24 or 48 hours.

Example 10

The following preservatives were individually added to samples ofrecombinant human thrombin DP, reconstituted with normal saline: phenol(1%, w/v), m-cresol (0.2%, v/v), methylparaben (0.5%, w/v),propylparaben (1%, w/v), or chlorobutanol (0.5%, w/v). Samples werestored at 25° C. Precipitation was observed in the phenol and m-cresolsamples after 24 hours. Samples containing 0.5% chlorobutanol remainedclear at 24 and 48 hours. Methylparaben and propylparaben were notsoluble in saline at the tested concentrations.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A pharmaceutical composition comprising: thrombin; abacteriostatically effective amount of a preservative selected from thegroup consisting of benzyl alcohol and chlorobutanol; and 0.10% to 5.0%(w/v) sucrose in aqueous solution.
 2. The composition of claim 1 furthercomprising one or more of a buffer, a salt, a polyol, a surfactant, anamino acid, or an additional carbohydrate.
 3. The composition of claim 1wherein the preservative is benzyl alcohol.
 4. The composition of claim3 wherein the benzyl alcohol is present at a concentration of 0.8%-1.5%(v/v).
 5. The composition of claim 1, further comprising mannitol. 6.The composition of claim 5 wherein the mannitol and sucrose are presentat a ratio of mannitol:sucrose greater than 1:1 but not greater than2.5:1 (w/w).
 7. The composition of claim 1 wherein the thrombin is humanthrombin.
 8. The composition of claim 7 wherein the human thrombin isrecombinant human thrombin.
 9. The composition of claim 1 wherein thethrombin is bovine thrombin.
 10. A composition comprising: thrombin; apharmaceutically acceptable buffer; 0.10% to 5% (w/v) sucrose; and apreservative selected from the group consisting of benzyl alcohol at aconcentration of 0.8% to 1.5% (v/v) or chlorobutanol at a concentrationof 0.4% to 0.6% (w/v), in aqueous solution at pH 5.7-7.4.
 11. Thecomposition of claim 10 wherein the preservative is benzyl alcohol. 12.The composition of claim 11 wherein the concentration of benzyl alcoholis 0.8% to 1.0% (v/v).
 13. The composition of claim 10 wherein thesucrose concentration is 0.5% to 3.0% (w/v).
 14. The composition ofclaim 10 wherein the sucrose concentration is about 1% (w/v).
 15. Thecomposition of claim 10 wherein the buffer is selected from the groupconsisting of histidine, citrate, phosphate, Tris, succinate, andacetate buffers.
 16. The composition of claim 10, further comprisingmannitol.
 17. The composition of claim 16 wherein the mannitol andsucrose are present at a ratio of mannitol:sucrose greater than 1:1 butnot greater than 2.5:1 (w/w).
 18. The composition of claim 10 whereinthe diluent comprises sodium chloride.
 19. The composition of claim 10wherein the thrombin is human thrombin.
 20. The composition of claim 19wherein the human thrombin is recombinant human thrombin.
 21. Thecomposition of claim 10 wherein the thrombin is bovine thrombin.
 22. Thecomposition of claim 10 wherein the thrombin is present at aconcentration of 0.1 mg/mL to 5.0 mg/mL.
 23. The composition of claim 22wherein the thrombin is present at a concentration of 0.3 mg/mL to 3.0mg/mL.
 24. An aqueous composition consisting essentially of: 0.03 mg/mLto 1.6 mg/mL thrombin; 0.17% to 1.3% (w/v) sucrose; 1.1% to 1.6% (w/v)mannitol; 0.8% to 2.0% (w/v) NaCl; 0-1.6 mM CaCl₂; 0.001% to 0.32% (w/v)of a surfactant or high-molecular-weight polyethylene glycol; apharmaceutically acceptable buffer; and a bacteriostatically effectiveamount of a preservative selected from the group consisting of benzylalcohol and chlorobutanol, in aqueous solution at pH 5.7-7.4, whereinconcentration of the buffer is selected to provide approximatelyphysiological pH upon application of the composition in a surgicalsetting and wherein the ratio of mannitol:sucrose is greater than 1:1but not greater than 2.5:1 (w/w).
 25. The composition of claim 24wherein the ratio of mannitol to sucrose is 1.33:1 (w/w).
 26. Thecomposition of claim 24 wherein the molar ratio of sucrose:thrombin isat least 700:1.
 27. The composition of claim 26 wherein the molar ratioof sucrose:thrombin is at least 2000:1.
 28. The composition of claim 24wherein the preservative is benzyl alcohol at a concentration of 0.8% to1.5% (v/v).
 29. A process for preparing a stabilized thrombin solutioncomprising: providing a lyophilized composition comprising thrombin anda quantity of sucrose sufficient to stabilize the protein in thepresence of a bacteriostatically effective amount of a preservativeselected from the group consisting of benzyl alcohol and chlorobutanol;providing a diluent comprising a bacteriostatically effective amount ofbenzyl alcohol or chlorobutanol in water; and combining the lyophilizedcomposition and the diluent to form a solution, wherein theconcentration of sucrose in the solution is from 0.10% to 5.0% (w/v).30. The process of claim 29 wherein the lyophilized composition furthercomprises a buffer, a salt, or a polyol.
 31. A process for preparing astabilized thrombin solution comprising: providing a lyophilizedcomposition comprising thrombin and at least one pharmaceuticallyacceptable excipient; and reconstituting the lyophilized composition ina diluent to provide a pharmaceutically acceptable thrombin solution,wherein the diluent is selected to provide in the solution a benzylalcohol concentration of 0.8% to 1.5% (v/v) and a sucrose concentrationof 0.10% to 5% (w/v).
 32. The process of claim 31 wherein the solutionfurther comprises mannitol.
 33. The process of claim 32 wherein themannitol and sucrose are present in the solution at a ratio ofmannitol:sucrose greater than 1:1 but not greater than 2.5:1 (w/w). 34.A kit comprising: a pharmaceutically acceptable diluent comprising abacteriostatically effective amount of benzyl alcohol or chlorobutanolin water in a first sealed container; and a lyophilized compositioncomprising thrombin and a quantity of sucrose in a second sealedcontainer, wherein the quantity of sucrose is selected to provide asucrose concentration of 0.10% to 5.0% (w/v) upon reconstitution of thelyophilized composition with the diluent.
 35. The kit of claim 34wherein the diluent further comprises NaCl.
 36. The kit of claim 35wherein the diluent is bacteriostatic saline.
 37. The kit of claim 34,further comprising means for transferring the diluent from the firstsealed container to the second sealed container.
 38. The kit of claim34, further comprising an instruction sheet.
 39. The kit of claim 34,further comprising an applicator device.
 40. The kit of claim 39,wherein the applicator device is a syringe or a sprayer.
 41. The kit ofclaim 34, wherein the first and second sealed containers are packaged ina third container.